Fottea, Olomouc, 12(2): 231–253, 2012 231

Genotypic diversity of Dictyosphaerium–morphospecies (Chlorellaceae, Trebouxiophyceae) in African inland waters, including the description of four new genera

Lothar Krienitz1*, Christina Bock1,2, Kiplagat Kotut3 & Thomas Pröschold4,5

1Leibniz–Institute of Freshwater Ecology and Inland Fisheries, Alte Fischerhütte 2, D–16775 Stechlin–Neuglobsow, Germany; *Corresponding author e–mail: krie@igb–berlin.de 2University of Essen, Faculty of Biology, D–45141 Essen, Germany 3Plant and Microbial Sciences Department of Kenyatta University Nairobi, PO Box 43844 GPO 00100 Nairobi, Kenya 4University of Vienna, Department of Limnology, Althanstr. 14, A–1090 Vienna, Austria 5University of Rostock, Department of Applied Ecology, Albert–Einstein–Str. 3, D–18059 Rostock, Germany

Abstract: Trebouxiophytes of the Dictyosphaerium–morphotype from inland waters of Africa were studied using a polyphasic approach of SSU and ITS rDNA phylogeny, secondary structure of the ITS and observations made with a light microscope. Although the morphological criteria for differentiating species and genera are scarce, the genetic diversity of these algae was very high. Based on our genetic analysis findings, we described four new genera containing five new species: Compactochlorella dohrmannii, Compactochlorella kochii, Kalenjinia gelatinosa, Marasphaerium gattermannii and Masaia oloidia. Diversity and distribution of Chlorella–related colonial chlorophytes in the tropical and temperate zones were compared and discussed.

Key words: Inland waters of Africa, Chlorella, Compactochlorella nov. gen., Dictyosphaerium, diversity, geographic distribution, green algae, Kalenjinia nov. gen., Marasphaerium nov. gen., Masaia nov. gen.

Introduction and recommended studies using a polyphasic approach combining morphological and molecular Coccoid green algae are among the most diverse phylogenetic methods. microphytes. In their famous handbook, Komárek Studies about algal biodiversity in Africa & Fott (1983) compiled more than 1200 taxa. have been hampered by several circumstances. For However, these taxa are defined by morphology. more than 100 years, algae in phytoplankton have To discover the real genotypic diversity of been investigated. However, a general limitation of coccoid green algae only few reports with limited this classical research, especially in the early years progress have been made. For example, Potter is that only fixed samples were taken by scientific et al. (1997) came up with a thesis of convergent travellers and later studied in various laboratories. evolution of morphology that masks the extensive These findings can be brought into agreement biodiversity among coccoid picoplankton. Fawley only with difficulties with the today’s views over et al. (2004) studied the molecular diversity of the systematics of algae. The second half of the coccoid and monadoid green algae in 20 lakes of 20th century saw an increase in the documentation North America and found 93 distinct SSU rRNA of detailed morphological characteristics leading genotypes, 89 of those were new to science. to the generation of data useful for comparative Fawley et al. (2005) evaluated the morphospecies studies. The most usable findings were published concept in Selenastraceae and concluded that on lakes of the southern and eastern Africa numerous cryptic species are hidden by one and (Huber–Pestalozzi 1929; Talling 1987; Cocquyt the same morphotype. Pröschold & Leliaert et al. 1993). However, the majority of systematic (2007), Coesel & Krienitz (2008) and Rindi et al. considerations on material from tropical countries (2010) discussed the state of the art in diversity are based on identification keys established for and taxonomic conceptions in green algae temperate zone taxa. Furthermore, investigations 232 Krienitz et al.: Genotypic diversity of Dictyosphaerium on living and cultured algal material including water chemistry or the strong interaction with sequence analyses are largely missing. Until now, consumer and decomposer populations in the only few cultured strains of coccoid green algae waters studied. The question that remains very from tropical Africa have been investigated by contentious is the amount of morphological modern systematic approaches (Luo et al. 2006, differences that are necessary to reflect the 2010; Bock et al. 2010, 2011a; Krienitz & Bock genotypic diversity of these Dictyosphaerium– 2011; Krienitz et al. 2010, 2011). like green algae. Molecular phylogenetic It is widely accepted that microbial diversity studies have shown that the Dictyosphaerium– differs fundamentally from biodiversity of larger morphotype evolved independently in different animals and plants (Norton et al. 1996). Some clades of the Chlorophyta (Bock et al. 2010, workers, including Fenchel & Finlay (2004), 2011b; Krienitz et al. 2010, 2011). Out of a have taken up the hypothesis of Baas–Becking collection of 27 chlorellacean strains isolated (1934) based on a metaphor by Beijerinck, from different inland waters of Africa, 24 strains which suggests that free–living microbes have a belonged to the Dictyosphaerium–morphotype. cosmopolitan distribution and that most protistan Based on these strains this study will address the organisms (microalgae and protozoa), smaller questions (i) how diverse are these coccoid green than one millimeter in size, have a worldwide algae in terms of morphology and phylogeny, distribution wherever their required habitats are and (ii) are the tropical Dictyosphaerium–like realised (“everything is everywhere, but, the algae genotypically identical with those from environment selects” – see De Wit & Bouvier the temperate climatic region? The outcome of 2006). This is a result of their ubiquitous dispersal this study is the description of four new genera driven by huge population sizes, and consequently including five new species. a low probability of local extinction. However, this hypothesis is only based on the phenotypic (“morphospecies”) approach and depends on Material and Methods clear identification of the microorganisms. For microalgae, the hypothesis is controversial and In this study 67 strains belonging to the class remains the subject of ongoing discussions Trebouxiophyceae were used (Table 1). Out of 28 (Coleman 2002; Finlay & Fenchel 2002; Foissner African strains, 26 strains were isolated by one 2006). Further studies, including more isolates of the authors from eight inland waters of Kenya, from different regions of the world, are needed and one or two waters of Angola, Tanzania, to prove or reject the hypothesis on the universal Tunisia, Uganda, and Zambia, respectively. These dispersal of microalgae. habitats were of different characteristics. Beside One of the most common morphotypes of the large Lake Victoria, smaller lakes of the Rift coccoid green algae in the phytoplankton of inland Valley, the lakes Baringo, Naivasha and Oloidien waters is represented by members of the genus were chosen as sampling area. Other strains were Dictyosphaerium. The genus is characterized isolated from ephemeral pools in the Ngorongoro by green spherical cells interconnected by Crater in Tanzania, Nakuru National Park in mucilaginous strands in colonies surrounded by Kenya and from a park pond in Nairobi. We also thick gelatinous envelopes (Fig. 1). Although this isolated strains from sewage oxidation ponds, morphotype has a world–wide distribution, it is such as the final sewage pond of the Nakuru town difficult to compare findings from different climatic sewage plant, and from a sewage pond on the regions. On the base of their morphology, about 11 Djerba island in Tunisia. Additional strains were species are known (Komárek & Perman 1978). The collected in rivers or water channels such as the type species Dictyosphaerium ehrenbergianum Mara river in the Masai Mara National Reserve Nägeli was found in European waters, and closely in Kenya, from the Kunene and Okawango in related morphotypes were also identified as very Angola, and from the Kazinga channel in Uganda, common in tropical inland waters. Microscopic which is connected to lakes Albert and George. studies have revealed that in comparison to the We isolated single Chlorella–like cells or phytoplankton from other continents, samples colonies of the Dictyosphaerium–morphotype from Africa appear to be especially rich in by glass–capillaries from the field samples and specimens with thick mucilaginous envelopes. transferred them to a liquid medium. All strains This unusual phenomenon may be a response to were maintained at the strain collection of the Fottea, Olomouc, 12(2): 231–253, 2012 233

IGB (Leibniz–Institute of Freshwater Ecology For the first phylogenetic tree, the SSU was and Inland Fisheries). The strains were grown manually aligned on the basis of the predicted in a modified Bourrelly medium (Hegewald et secondary structure model for Micractinium al. 1994; Krienitz & Wirth 2006) on agar at 15 pusillum (Luo et al. 2006). Stems and loop regions °C or in suspensions at room temperature under of the 18S of the strains were aligned to each other a 14 h : 10 h light–dark regime. New strains were respectively. The ITS regions were more difficult deposited at the Culture Collection of Algae and to align due to a high degree of divergence between Protozoa (CCAP, Oban, UK). The algae were the sequences. Within this regions, we aligned investigated using a Nikon Eclipse E600 light them strictly according to their predicted secondary microscope with differential interference contrast. structure, e.g. stem regions were aligned separately Microphotographs were taken with a Nikon from loops and unmatching regions. Dubiously digital camera DS–Fi1, and Nikon software NIS– aligned regions were excluded. The phylogenetic Elements D (Nikon Corporation, Tokyo, Japan). tree was inferred by maximum likelihood settings In this study, we sequenced the SSU and ITS on a partitioned data set using Treefinder (Jobb rRNA gene sequences of 13 strains to establish new 2008). Models for each partition, as proposed by sequence data, which were submitted to GenBank. Treefinder under AICc criterion, were as followed: Additionally, 57 sequences from GenBank were 18S (J2:G:5 model, 1797 bases), ITS1 (J1:G:5 included in this data set (Table 1). model, 408 bases), 5.8S (HKY model, 141 bases), Genomic DNA was extracted using a ITS2 (GTR:G:5 mode, 347 bases). To test the lysozym/sodium phosphate method. The algal confidence of the tree topology, bootstrap analyses cells were mechanical disrupted with glass beads were calculated by distance (neighbor–joining; using the TissuelyserII (Qiagen GmbH, Hilden, NJ; 1000 replicates) and maximum parsimony Germany) in the presence of 600 µl sodium (MP; 1000 replicates; with heuristic search phosphate buffer (120 mM) and 100 µl SDS options based on simple taxon addition, tree– (25%). After centrifugation for 6 min, the liquid bisection–reconnection (TBR) branch swapping phase was transferred to a clean reaction tube algorithm and Multrees option enabled) using and incubated with 200 µl Lysozym at 37 °C for PAUP*, portable version 4.0b10 (Swofford 2002) 1 hour. Afterwards, the probes were incubated at and maximum likelihood criteria using Treefinder 55 °C over night after adding 150 µl SDS (25%) (ML; 1000 replicates; settings as described above). and 12.5 µl proteinase K. Protein precipitation The Bayesian inference (MB) was calculated was done by adding 7.5 M ammonium acetate using MrBayes version 3.1 (Huelsenbeck & (0.4 times of the existing volume) and incubating Ronquist 2001). Two runs with four chains of on ice for 5 min. The supernatant was transferred Markov chain Monte Carlo (MCMC) iterations to a clean reaction tube after a centrifugation step were performed with tree sampling every 100 and DNA was purified with 0.7% isopropanol and generations. The model GTR+I+G with gamma centrifugation for 1hour. The liquid was discarded shape parameter and proportion of invariable sites and DNA pellet was washed with ethanol (80%). was used for each partition. The parameters were The SSU and ITS rRNA genes were amplified and unlinked and allowed to vary across the partitions. sequenced as described by Bock et al. (2011a). The stationary distribution was assumed after For the phylogenetic analyses, a data 2,000,000 generations when the average standard set of 68 taxa with 2693 aligned base positions deviations of split frequencies between two runs and a dataset with 71 taxa and 2815 bases were were below 0.01. The first 25% of the calculated used respectively. In order to obtain an adequate trees were discarded as burn–in. A 50% majority– representation of chlorellacean algae, different rule consensus tree was calculated for posterior sequences were selected according to Bock et probabilities (PP). al. (2011a, b) with Catena viridis as outgroup in For the second phylogenetic tree, the addition to the newly sequenced strains. The two sequences were initially aligned using the phylogenetic trees presented in our results were ClustalW algorithm integrated in SOAP v.1.2 inferred using two different ways of alignment: alpha 4 (Loytynoja & Milinkovitch 2001). The manual alignment according to the secondary stability of the alignment was assessed using SOAP structure; and the ClustalW algorithm integrated in by comparison of different ClustalW alignments SOAP v.1.2 alpha 4 (Loytynoja & Milinkovitch using gap penalties from 7 to 20 by steps of 2.5 2001). and extension penalties from 2 to 10 by steps 234 Table 1. Strains used in this study. The strains are grouped according to the topology in the phylogram (Fig. 8). Authentic strains in bold letters.

Strain Strain- Species Origin Accession number Reference designation number in (SSU + ITS) the IGB- Collection b)

Chlorella- clade a) CCAP 211/81 Chlorella vulgaris Germany, saline pond Salzteich, Trebbichau, Sachsen-Anhalt FM205854 Luo et al. 2010

ACOI 856 C. pituita Portugal, Serra da Estrela FM205856 Luo et al. 2010

SAG 37.88 C. lobophora Russia, soil from forest, Krasnyj Rog, Brianskaia district FM205833 Luo et al. 2010

CCAP 211/84 C. variabilis USA, endosymbiont of Paramecium, NC84A AB206549 Hoshina et al. 2005

SAG 211-6 C. variabilis USA, endosymbiont of Paramecium, J.B. Loefer, 1934 FM205849 Luo et al. 2010

CCAP 260/11 KR 2007/5 C. rotunda Angola, River Okawango near Mutango HQ111433 Bock et al. 2011a

SAG 3.83 C. heliozoae Canada, endosymbiont in Acanthocystis, Newfoundland, bog FM205850 Luo et al. 2010

pool in Terra Nova National Park K rienitz SAG 222-2a C. pulchelloides France, near Amiens, E.A. George FM205857 Luo et al. 2010

CCAP 211/118 CB 2008/50 C. pulchelloides Germany, Lake Feldberger Haussee, Mecklenburg-Western HQ111431 Bock et al. 2011a et al.: Genotypic diversity of Pomerania

CCAP 211/116 CB 2008/110 C. chlorelloides Germany, Lake Pragsdorfer See, Mecklenburg-Western HQ111432 Bock et al. 2011a Pomerania

UTEX 938 C. colonialis USA, unknown FM205862 Luo et al. 2010

CCAP 211/119 CB 2008/73 C. singularis Kenya, Sewagepond Nakuru HQ111435 Bock et al. 2011a

CCAP 222/18 Wolf 2000/1 C. elongata Germany, Berlin, Steglitz, fountain-pool FM205858 Luo et al. 2010

ock CCAP 211/120 CB 2008/69 C. volutis Kenya, Rhinopool, Nakuru National Park HQ111434 B et al. 2011a Dictyosphaerium

CCALA 260 C. sorokiniana Slovakia, Piestany, thermal spring FM205860 Luo et al. 2010

CCAP 211/8k C. sorokiniana USA, University, Austin, Texas, Waller Creek FM205859 Luo et al. 2010 Fottea, Olomouc, 12(2):231–253,2012235 Table 1. Cont.

CCAP 211/90 C. lewinii Chile, soil from the edge of a permanent freshwater pond in FM205861 Luo et al. 2010 a crater, Easter Island, R.A. Lewin

CCAP 222/71 CB 2007/25 Hindakia tetrachotoma Germany, Lake Pragsdorfer See, Mecklenburg-Western GQ487228 Bock et al. 2010 Pomerania

CCAP 222/73 CB 2007/27 „ Germany, Waldstich Zehdenick, Brandenburg GQ487231 Bock et al. 2010

CCAP 222/81 CB 2008/70 „ Kenya, Lake Baringo GQ487230 Bock et al. 2010

CCAP 222/82 CB 2008/71 „ Kenya, Lake Baringo GQ487232 Bock et al. 2010

CCAP 222/80 CB 2008/48 „ Germany, Lake Kleiner Tietzensee, Brandenburg GQ487233 Bock et al. 2010

CCAP 222/29 KR 2006/317 Hindakia fallax Kenya, Lake Victoria, Dunga GQ487223 Bock et al. 2010

CCAP 222/30 KR 2006/320 „ Kenya, Lake Victoria, Dunga GQ487224 Bock et al. 2010

CCAP 222/47 KR 2007/12 Heynigia riparia Angola, River Kunene near Epupa Falls GQ487225 Bock et al. 2010

CCAP 222/2D Heynigia UK, Windermere, Cumbria GQ487221 Bock et al. 2010 dictyosphaerioides

CCAP 248/5 Micractinium pusillum Kenya, Sewagepond, Nakuru FM205836 Luo et al. 2010

SAG 42.98 M. belenophorum Germany, Berlin, Lake Lietzensee FM205879 Luo et al. 2010

SAG 30.92 Didymogenes palatina Germany, water tank, Jülich, E. Hegewald 1982/83 FM205840 Luo et al. 2010

SAG 18.91 Didymogenes anomala Germany, River Rhein near Linz, E. Hegewald, 1990/11 FM205839 Luo et al. 2010

SAG 2015 KR 1996/4 Actinastrum hantzschii Germany, River Elbe near Aken, Sachsen-Anhalt FM205841 Luo et al. 2010

CCMP 2446 Meyerella planctonica USA, Lake Itasca, Minnesota AY543040 Fawley et al. 2005 AY543045

Parachlorella- clade CCAP 222/20 KR 2006/302 Dictyosphaerium Kenya, Lake Naivasha GQ487192 Bock et al. 2011b ehrenbergianum

CCAP 222/21 KR 2006/303 „ Kenya, Lake Naivasha GQ487193 Bock et al. 2011b

CCAP 222/26 KR 2006/311 „ Kenya, Lake Naivasha GQ487195 Bock et al. 2011b 236 Table 1. Cont.

CCAP 222/22 KR 2006/304 „ Kenya, Lake Naivasha GQ487194 Bock et al. 2011b CCAP 222/27 KR 2006/312 „ Kenya, Lake Naivasha GQ477062 Bock et al. 2011b

UTEX 75 „ UK, pond near Cambridge, E.G. Pringsheim, 1940 GQ176856 Krienitz et al. 2010

CCAP 222/1A „ UK, pond near Cambridge, E.G. Pringsheim, 1940 GQ176861 Krienitz et al. 2010

CCAP 222/10 LW 2003/183 „ Germany, Lake Geron, Gransee GQ176857 Krienitz et al. 2010

CCAP 222/23 KR 2006/305 „ Kenya, Lake Victoria, Homa Bay GQ176859 Krienitz et al. 2010

CCAP 222/14 KR 2006/31 „ Tunisia, oxidationpond, Jerba GQ176858 Krienitz et al. 2010

CCAP 222/92 CB 2008/76 D. libertatis Kenya, Uhuru-Pond Nairobi GQ487211 Bock et al. 2011b

Bock et al. 2011b Dictyosphaerium CCAP 222/85 CB 2008/54 Germany, Oxidationpond Neuglobsow, Brandenburg, GQ487206 lacustre SAG 2046 KR 1999/1 Parachlorella Germany, Nonnenbach brook, a tributary of Lake Tollense, FM205845 Luo et al. 2010 beijerinckii Mecklenburg-Western Pomerania

SAG 211-11g Parachlorella kessleri USA, New York, M. Winokur, 1945 FM205885 Luo et al. 2010

K

CCAP 222/25 KR 2006/310 Dictyosphaerium Uganda, Kazinga-Channel GQ176862 Krienitz et al. 2010 rienitz morphotype

CCAP 222/24 KR 2006/309 Marasphaerium Kenya, River Mara GQ477057 this study et al.: Genotypic diversity of gattermannii Heg. 1983/17 Marasphaerium Germany, Berlin, Lake Stölpchensee HQ322127 this study gattermannii

ACOI 1988 Dictyosphaerium unknown GQ176863 Krienitz et al. 2010 morphotype

CCAP 222/1C Dictyosphaerium UK Windermere, Cumbria, G. Jaworski GQ176864 Krienitz et al. 2010 morphotype Dictyosphaerium

CCAP 222/93 CB 2008/19 Mucidosphaerium Germany, Lake Wollingster See, Lower Saxony GQ487218 Bock et al. 2011b sphagnale Fottea, Olomouc, 12(2):231–253,2012237 Table 1. Cont.

CCAP 222/96 CB 2008/6 Mucidosphaerium Germany, pond Klipsmoorheide, Lower Saxony GQ487216 Bock et al. 2011b palustris

UTEX 731 Mucidosphaerium Canada, Nova Scotia, R.A. Lewin 1952 GQ176861 Krienitz et al. 2010 pulchellum

ACOI 1719 Mucidosphaerium Portugal, Abrantes, Campo Militar de Sta Margarida, GQ487201 Bock et al. 2011b planctonicum Barragem do Monte Novo CCAP 222/61 CB 2007/8 Compactochlorella Germany, Lake Jabeler See, Mecklenburg-Western HQ322125 this study kochii Pomerania CB 2008/47 Compactochlorella Germany, Lake Grosser Tietzensee near Rheinsberg, HQ322124 this study kochii Brandenburg CB 2008/104 Compactochlorella Kenya, Rhinopool Nakuru National Park HQ322126 this study kochii CCAP 222/7 LW 2003/37 Compactochlorella Tanzania, ephemeral Yellow-Bill-Pool, Ngorongoro Crater GQ487244 this study kochii CCAP 222/5 KR 2002/24 Compactochlorella Kenya, Sewagepond, Nakuru GQ477058 this study dohrmannii CCAP 211/85 KR 2003/30 Masaia oloidia Kenya, Lake Oloidien, cruci GQ477059 this study CCAP 222/32 KR 2006/322 Masaia oloidia Kenya, Lake Oloidien, cruci GQ477060 this study CB 2008/72 Masaia oloidia Kenya, Sewagepond, Nakuru HQ322128 this study

SAG 11.86 Closteriopsis acicularis Germany, Lake Grömitzer See, E. Hegewald 1977/126 FM205847 Luo et al. 2010

CCAP 222/43 KR 2007/3 Dictyosphaerium Angola, River Okawango near Mutango GQ477066 this study morphotype CB 2008/94 Kalenjinia gelatinosa Kenya, Sewagepond, Nakuru HQ322129 this study CCAP 222/8 LW 2003/125 Kalenjinia gelatinosa Kenya, Sewagepond, Nakuru GQ477061 this study

SAG 41.98 Dicloster acuatus Ukraine, pond at Krasne FM205848 Luo et al. 2010 238 Table 1. Cont.

Outgroup SAG 65.94 KR 1991/4 Catena viridis Germany, pond Oxidationteich Neuglobsow, Brandenburg GU592792 Bock et al. 2010

CCAP 283/1 Hegewaldia parvula USA, Lemoncove, California FM205842 Pröschold et al. 2010 CCAP 283/2 Hegewaldia parvula USA, Lemoncove, California FM205843 Pröschold et al. 2010

a) Abbreviations: ACOI = Coimbra Collection of Algae, Portugal CAUP = Culture Collection of Algae of Charles University in Prague, Czech Republic CCALA = Culture Collection of Autotrophic Organisms, Trebon, Czech Republic CCAP = Culture Collection of Algae and Protozoa, Oban, UK CCMP = The Provasoli-Guillard National Center for Culture of Marine Phytoplankton, USA SAG = Sammlung von Algenkulturen der Universität Göttingen, Germany UTEX = The Culture Collection of Algae at The University of Texas at Austin, USA b) For own isolates the initials of the isolator were given: CB = Christina Bock, KR = Lothar Krienitz, LW = Luo Wei

compensatory base changes (CBCs) according to hemi–compensatory base changes(h–CBCs)and nonhomoplasious synapomorphies (NHS), H M et al. 2011b; ehrenbergianum structure as template ( constructed with the help of mfold with the ITS2 (GTR:G:5mode,399bases). model, 470bases),5.8S(HKY model,139bases), 18S (GTR:G:5model, 1807 bases), ITS1 (J1:G:5 interfered as describedabovewiththeML models: and thecorrespondingbootstrapvalueswere in 2815alligned bases. The phylogenetic tree computing a 90%consensusalignment, resulting of 1.5.Regionsinstability were excluded by the the cells at their apical end. In contrast, cells of Hindakia (Fig.6),themucilaginous stalks attached of of freshlyinoculatedsuspensioncultures. agar–cultures or occasionally during the first days collection. Colonies werebestvisible onfresh maintenance under the conditions of the strain the colonial life form disappeared after longer of the showed moreorlessthecharacteristic colonies 6 and7),somestrainsbecame solitary butmost (Figs 4and5).Underculture conditions (Figs at the apical (Fig. 1) or at the longitudinal side within the colony. The stalks attached the cells depending on thenumber of cells joined together cross–shaped (Fig.2)ormorecomplex (Fig. 3) after liberation of theautosporesweresimple, established bymothercellwallremnants The interconnecting strands between the cells, in diameter, varied from spherical to ovoid. of variability. The cells, whichwere3–14µm colony organization a certainamount showed Cell shapeandsizeseveralfeaturesof chloroplast carriedastarch–sheathed pyrenoid. identical to Chlorella was cells the of anatomy inner The identifiable. colonies mucilaginous envelopes (Fig. 1) were clearly 1–5), (Figs with conditions field Under Results Structures weredrawnbyPseudoViewer ( K an rienitz arin Chlorella, suchasthe members ofthegenus 2006). Parachlorella Dictyosphaerium–morphotype covered by The ITS2secondarystructure was In strains belonging to the close relatives etal.(2003)and Dictyosphaerium–morphotype. Generally,

et al.: Genotypic diversity of K rienitz /Dictyosphaerium–relationship : theparietal, cup–shaped et al. 2010) tolocate C oleman Dictyosphaerium (2003,2007). B yun B ock

D. &

Fottea, Olomouc, 12(2): 231–253, 2012 239

Table 2. Comparison of CBCs and hemi-CBCs within the ITS2 between the newly erected species.

Helix I Helix II Helix III Helix IV CBC/ hemi- CBC/ hemi- CBC/ hemi-CBC CBC/ hemi-CBC CBC CBC Marasphaerium gattermannii – / – 5 / – – / 2 6 / – vs Compactochlorella kochii

Compactochlorella kochii vs – / – 2 / 1 1 / 2 1 / 1 Compactochlorella dohrmannii

Compactochlorella dohrmannii 1 / 1 3 / – – / 2 1 / 1 vs Masaia oloidia

Masaia oloidia vs – / – 2 / 1 1 / 3 3 / – Kalenjinia gelatinosa

were attached by the stalks at their longitudinal a cluster containing the strain CCAP 222/24 from end (Fig. 7). the Mara river in Kenya, which is described as Three of the African strains of Chlorellaceae Marasphaerium gattermannii gen. et sp. nov. were solitary and belonged unambiguously to the in this paper. Next to these lineages evolved genus Chlorella. The 24 colony–forming strains members of the genera Mucidosphaerium and a of Dictyosphaerium–morphotype from African cluster containing three African strains described waters evolved in seven different lineages of below as Compactochlorella dohrmannii and Chlorellaceae (Figs 8 and 9). Four strains, C. kochii gen. and sp. nov. From C. kochii four from the lakes Victoria and Baringo, belonged strains were analysed, two of them from Africa to the genus Hindakia within the Chlorella– and two from Germany. Unfortunately, the DNA clade. The other strains occurred in different sequence from the African strain in the culture clusters of the Parachlorella–clade. Whereas collection (CCAP 222/7) was imprecise at the end the sister–relationship of the Chlorella– and the of the ITS2 and therefore we selected the cultured Parachlorella–clade was supported sufficiently, strain CCAP 222/61 from Germany as authentic the general topology of the lineages within these strain those sequence was complete. Adjacent to two clades was not supported by our analyses. these lineages, three strains from Kenya’s standing However, the position of the genus Hegewaldia waters, which are newly described here as Masaia previously assigned to the Chlorella–clade is oloidia gen. et sp. nov., established its own cluster. unclear (Fig. 9). The strain CCAP 222/43 exhibited typical colonies Eight African isolates belonged to the genus of a Dictyosphaerium–morphotype and clustered Dictyosphaerium, seven of them were designated together with the needle–shaped Closteriopsis to the type species D. ehrenbergianum Nägeli, and acicularis (G.M. Smith) Belcher et Swale. the other species was determined as D. libertatis Furthermore, two new spherical and colonial C. Bock, Pröschold et Krienitz. Closely related to strains from the Nakuru sewage oxidation pond, these two clusters is the strain CCAP 222/25 from described here as Kalenjinia gelatinosa gen. et Uganda, which will be studied in more detail by sp. nov., established a lineage placed between Pavel Škaloud and his team. Two European strains Closteriopsis and the colonial needle–shaped (ACOI 1988 and CCAP 222/1C) were related to Dicloster acuatus Jao, Wei et Hu. 240 Krienitz et al.: Genotypic diversity of Dictyosphaerium

Figs 1 – 5. The Dictyosphaerium–morphotype in field samples from Lake Victoria (Figs 1–3) and a sewage pond at Nakuru (Figs 4, 5). The empty arrowhead indicates the interconnecting strands between the cells. (1) spherical colony covered by a thick mucilaginous envelope which is made visible by silt particles and picoplanktonic cyanobacteria. (2) colony with two–, or four–celled mother cells and cross–shaped interconnecting strands; (3) articulated strands of a colony of mother cells which already liberated the autospores (only two autospores remained in the upper left mother cell). Scale bars 10 µm.

The topology of the phylogenetic trees bootstrap values of the lineages. recovered from two different alignments (manual To evaluate the newly erected five species alignment, Fig. 8, and alignment computed with according to the CBC concept, we compared ClustelW, Fig. 9) was in general congruent. the number of CBCs and hemi–CBCs and No major discrepancies occurred between the found a remarkable number of differences different alignment methods; differences were among the taxa (for details see Fig. 10, and only observed in the placements of lineages that Table 2). Marasphaerium gattermannii and received no statistical support in either method. Compactochlorella kochii differ in 11 CBCs and 2 Within the Chlorella–clade, the cluster with hemi–CBCs with additional base pairs in all helices sequences of Micractinium and Didymogenes within the ITS2. The two species belonging to the exchanged position with the Hindakia/Heynigia new genus Compactochlorella differ in 4 CBCs cluster. Within the Parachlorella–clade, the cluster and 4 hemi–CBCs and additional base pairs in containing Marasphaerium, Mucidosphaerium, Helices I, II and IV. Considerable CBCs occurred Compactochlorella and ACOI 1988/CCAP222/1C between the genera Compactochlorella and (Fig. 8) is separated in different lineages (Fig. 9). Masaia (5 CBCs and 4 hemi–CBCs) and between Small differences were also observed within the Masaia and Kalenjinia (6 CBCs; 4 hemi–CBCs). Fottea, Olomouc, 12(2): 231–253, 2012 241

Marasphaerium Krienitz, C. Bock, Kotut et Pröschold gen. nov. Latin diagnosis: Cellulae viridis, sphaericae, planctonicae. Chloroplastus unicus, parietalis, poculiformis, pyrenoide granis amylis tecto. Reproductio asexualis autosporum ope, reproductio sexualis ignota. Cellulae solitariae vel in coloniis, 2–4 cellularis, interdum tegumento gelatinoso vestitae. A generibus ceteris familiae ordine nucleotidorum in 18S rDNA et ITS differt.

Cells green, spherical, planktonic. Single cup– shaped chloroplast with starch–covered pyrenoid. Asexual reproduction by autosporulation, sexual reproduction not observed. Cells solitary or in colonies of 2–4 cells, covered by a gelatinous envelope. Genus differs from other genera of the family by the order of the nucleotides in SSU and ITS rRNA gene sequences. Typus generis: Marasphaerium gattermannii Krienitz, C. Bock, Kotut et Pröschold sp. nov. Etymology: the genus is named according to its locus classicus, the Mara river in the Masai Mara National Reserve, Kenya.

Marasphaerium gattermannii Krienitz, C. Bock, Kotut et Pröschold sp. nov. Latin diagnosis: Cellulae solitariae vel in coloniis, planctonicae, interdum tegumento gelatinoso vestitae. Coloniae parvae, 2–4 cellularis, cellulis funibus subtilibus hyalinis iunctis. Cellulae sphericae, raro late ovalis, 4–13 µm in diametro. Chloroplastus unicus, parietalis, poculiformis, pyrenoide granis amylis tecto. Reproductio asexualis autosporum ope. A speciebus ceteris generis ordine nucleotidorum in 18S rDNA et ITS differt. Figs 6, 7. The Dictyosphaerium–morphotype in culture. The black arrows indicate the place of attachment of the interconnecting strands to the cell wall. (6) colony of Hindakia Cells solitary or in colonies, planktonic, covered fallax (CCAP 222/29), a member of the Chlorella–clade; by a gelatinous envelope. Colonies small, 2–4 the strands are attached to the apical cell side; (7) colony celled, cells connected by hyaline mother cell of Dictyosphaerium ehrenbergianum (CCAP 222/27), a member of the Parachlorella–clade; the strands are attached wall remnants. Cells spherical, seldom broad to the longitudinal cell side. Scale bars 10 µm. oval, 4–13 µm in diameter. Single cup–shaped chloroplast with starch–covered pyrenoid. Asexual reproduction by autosporulation. Differs from species of other genera by the order of To align the phylogenetic findings with the the nucleotides in SSU and ITS rRNA gene morphology features, the five authentic strains of sequences. the new taxa were subjected again to microscopic Holotype: material of the authentic strain CCAP investigations. Micrographs of these authentic 222/24 is cryopreserved in metabolic inactive state strains are given in Figs 11–15, whereas drawings at the Culture Collection of Algae and Protozoa, including the iconotypes are shown in Figs 16–20. Oban, Scotland. Results of the comparison of their morphological Isotype: an air–dried as well as a formaldehyde– features are provided in Table 3. fixed sample of the authentic strain CCAP 222/24, Generic and species descriptions deposited at the Botanical Museum at Berlin– 242 Krienitz et al.: Genotypic diversity of Dictyosphaerium

Dahlem under the designation B400040739. Type locality: Mara river in the Masai Mara National Reserve, Kenya. Etymology: The species is named in memory of the late Rolf Gattermann, who was a leading authority in Zoology and behavioural biology of mammals. He was impressed by the wildlife in East Africa and inspired the first author to study African nature. Authentic strain: CCAP 222/24. Iconotype: Figure 16*

Compactochlorella Krienitz, C. Bock, Kotut et

Pröschold gen. nov. Colonies 2 or 4 cells, very unstable With 2 or 4 cells, developing lumpy With stage complexes of colonies different 2 or 4 cells, sometimes cells are closely With attached to tetrads 2, 4 or 8 cells With 2 or 4 cells With Latin diagnosis: Cellulae viridis, sphaericae, planctonicae. Chloroplastus unicus, parietalis, poculiformis, pyrenoide granis amylis tecto. Reproductio asexualis autosporum ope, reproductio sexualis ignota. Cellulae solitariae vel in coloniis, 2–4 cellularis, vel in aggregationibus compactis, interdum tegumento gelatinoso vestitae. A generibus ceteris familiae ordine nucleotidorum in 18S rDNA et ITS differt.

Cells green, spherical, planktonic. Single cup– shaped chloroplast with starch–covered pyrenoid. Asexual reproduction by autosporulation, sexual reproduction not observed. Cells solitary or in Mother Cells Spherical, 9–13 µm, with 2, 4 or 8 autospores Spherical, rarely oval, 8–12 µm, with 2, or 4 autospores Spherical, rarely oval, 7–12 µm, with 2 or 4 autospores Spherical, rarely oval, 8–12 µm, with 2, 4 or 8 autospores, Spherical, rarely oval, 9–14 µm, with 2 or 4 autospores colonies of 2–4 cells, or in compact aggregations, covered by a gelatinous envelope. Genus differs from other genera of the family by the order of the nucleotides in SSU and ITS rRNA gene sequences. Typus generis: Compactochlorella kochii Krienitz, C. Bock, Kotut et Pröschold sp. nov. Etymology: The genus is named according to the Vegetative Cells Vegetative Spherical, 5–9 µm Spherical or oval, 5.5–8.5 µm Spherical or oval, 5–7.5 µm Spherical or oval, 5.5–8 µm Spherical or oval, 6–9 µm compact aggregations which were often developed by the colonies of the type species.

Compactochlorella kochii Krienitz, C. Bock, Kotut et Pröschold sp. nov. Latin diagnosis: Cellulae solitariae vel in coloniis, 2–4 cellularis, vel in aggregationibus compactis, interdum tegumento gelatinoso vestitae, planctonicae. Cellulis funibus subtilis hyalinis iunctis. Cellulae ovoides, Spherical, rarely broad oval, 4–6 µm Ovoid, 3–5 × 2.5–4 µm Ovoid, 3–4.5 × 2.5–3 µm Ovoid, 3.5–6 × 3–5 µm Ovoid or drop–shaped, 4–6 × 3.5–5.5 µm ovalis vel sphericae, 3–12 × 2.5–12 µm. Chloroplastus Autospores unicus, parietalis, poculiformis, pyrenoide granis amylis tecto. Reproduction asexualis autosporum ope. A speciebus ceteris generis ordine nucleotidorum in 18S rDNA et ITS differt.

Cells solitary or in 2–4 celled colonies, or in compact aggregations, covered by a gelatinous envelope, planktonic. Cells connected by hyaline Marasphaerium gattermannii 222/24 CCAP Compactochlorella kochii 222/61 CCAP Compactochlorella dohrmannii 222/5 CCAP Masaia oloidia 222/32 CCAP Kalenjinia gelatinosa 222/8 CCAP Species stalks. Cells ovoid, oval or spherical, 3–12 × 3. Morphological characteristics of the species newly described in this study. Table Fottea, Olomouc, 12(2): 231–253, 2012 243

Fig. 8. Maximum likelihood phylogram of the Chlorellaceae with Catena viridis as outgroup inferred from a concatenated set of SSU and ITS sequences. The phylogram is based on a partitioned dataset with manually aligned sequences according to their secondary structure. Hyphens correspond to values below 50 for BP and below 0.95 for PP. African strains in bold. Scale bar indicates substitutions per site. 244 Krienitz et al.: Genotypic diversity of Dictyosphaerium

Fig. 9. Maximum likelihood phylogram of the Chlorellaceae with Catena viridis as outgroup inferred from a concatenated set of SSU and ITS sequences. The phylogram is based on a partitioned dataset of a 90% consensus alignment predicted by SOAP (Loytynoja & Milinkovitch 2001) under different gap/extension penalties. Hyphens correspond to values below 50 for BP and below 0.95 for PP. African strains in bold. Scale bar indicates substitutions per site. Fottea, Olomouc, 12(2): 231–253, 2012 245

2.5–12 µm. Single cup–shaped chloroplast with the first author to study microphytes. starch–covered pyrenoid. Asexual reproduction Authentic strain: CCAP 222/5. by autosporulation. Differs from other species of Iconotype: Figure 18* the genus by the order of the nucleotides in SSU and ITS rRNA gene sequences. Masaia Krienitz, C. Bock, Kotut et Pröschold Holotype: material of the authentic strain CCAP gen. nov. 222/61 is cryopreserved in metabolic inactive state Latin diagnosis: Cellulae viridis, sphaericae, at the Culture Collection of Algae and Protozoa, planctonicae. Chloroplastus unicus, parietalis, pocu- Oban, Scotland. liformis, pyrenoide granis amylis tecto. Reproductio Isotype: an air–dried as well as a formaldehyde– asexualis autosporum ope, reproductio sexualis ignota. fixed sample of the authentic strain CCAP 222/61, Cellulae solitariae vel in coloniis, 2–4–8 cellularis, deposited at the Botanical Museum at Berlin– interdum tegumento gelatinoso vestitae. A generibus ceteris familiae ordine nucleotidorum in 18S rDNA et Dahlem under the designation B40004040. ITS differt. Type locality: lake Jabeler See, Mecklenburg– Western Pomerania, Germany. Cells green, spherical, planktonic. Single cup– Etymology: the species is named in honour of shaped chloroplast with starch–covered pyrenoid. Frank Koch, an authority in entomology. He Asexual reproduction by autosporulation, sexual makes frequent work visits to Africa, and inspired reproduction not observed. Cells solitary or in the first author to study African nature. colonies of 2–4–8 cells, covered by a gelatinous Authentic strain: CCAP 222/61. Iconotype: Figure 17* envelope. Genus differs from other genera of the family by the order of the nucleotides in SSU and ITS rRNA gene sequences. Compactochlorella dohrmannii Krienitz, C. Typus generis: Masaia oloidia Krienitz, C. Bock, Bock, Kotut et Pröschold sp. nov. Latin diagnosis: Cellulae planctonicae, solitariae vel in Kotut et Pröschold sp. nov. coloniis, 2–4 cellularis, interdum tegumento gelatinoso Etymology: the genus is named after the Masai, a vestitae. Cellulis funibus subtilis hyalinis iunctis. famous tribe living in East Africa Cellulae ovoides, ovalis vel sphericae, 3–12 × 2.5–10 µm. Chloroplastus unicus, parietalis, poculiformis, Masaia oloidia Krienitz, C. Bock, Kotut et pyrenoide granis amylis tecto. Reproductio asexualis Pröschold sp. nov. autosporum ope. A speciebus ceteris generis ordine Latin diagnosis: Cellulae solitariae vel in coloniis, nucleotidorum in 18S rDNA et ITS differt. 2–4–8 cellularis, interdum tegumento gelatinoso vestitae, planctonicae. Cellulis funibus subtilis Cells solitary or in 2–4 celled colonies, or in hyalinis iunctis. Cellulae ovoides, ovalis vel sphericae, compact aggregations, covered by a gelatinous 3.5–12 × 3–12 µm. Chloroplastus unicus, parietalis, envelope, planktonic. Cells connected by hyaline poculiformis, pyrenoide granis amylis tecto. stalks. Cells ovoid, oval or spherical, 3–12 × Reproductio asexualis autosporum ope. A speciebus 2.5–10 µm. Single cup–shaped chloroplast with ceteris generis ordine nucleotidorum in 18S rDNA et starch–covered pyrenoid. Asexual reproduction ITS differt. by autosporulation. Differs from other species of the genus by the order of the nucleotides in SSU Cells solitary or in 2–4–8 celled colonies, covered and ITS rRNA gene sequences. by a gelatinous envelope, planktonic. Cells Holotype: material of the authentic strain CCAP connected by hyaline stalks. Cells ovoid, oval 222/5 is cryopreserved in metabolic inactive state or spherical, 3.5–12 × 3–12 µm. Single cup– at the Culture Collection of Algae and Protozoa, shaped chloroplast with starch–covered pyrenoid. Oban, Scotland. Asexual reproduction by autosporulation. Differs Isotype: an air–dried as well as a formaldehyde– from species of other genera by the order of fixed sample of the authentic strain CCAP 222/5, the nucleotides in SSU and ITS rRNA gene deposited at the Botanical Museum at Berlin– sequences. Dahlem under the designation B40004041. Holotype: material of the authentic strain CCAP Type locality: sewage oxidation pond, Nakuru, 222/32 is cryopreserved in metabolic inactive state Kenya. at the Culture Collection of Algae and Protozoa, Etymology: the species is named in honour of Oban, Scotland. Klaus Dohrmann, a microbiologist. He inspired Isotype: an air–dried as well as a formaldehyde– 246 Krienitz et al.: Genotypic diversity of Dictyosphaerium

Fig. 10. Comparison of the secondary structure of the helices I – IV of ITS2 rRNA gene of authentic strains of Marasphaerium gattermannii (CCAP 222/24), Compactochlorella kochii (CCAP 222/61), Compactochlorella dohrmannii (CCAP 222/5), Masaia oloidia (CCAP 222/32) and Kalenjinia gelatinosa (CCAP 222/8). The number of differences is given in Table 2. Fottea, Olomouc, 12(2): 231–253, 2012 247

Figs 11 – 15. Microphotographs of the newly described genera and species: (11) Marasphaerium gattermannii; (12) Compactochlorella kochii; (13) Compactochlorella dohrmannii; (14) Masaia oloidia; (15) Kalenjinia gelatinosa. Scale bars 10 µm. fixed sample of the authentic strain CCAP 222/32, rDNA et ITS differt. deposited at the Botanical Museum at Berlin– Dahlem under the designation B40004042. Cells green, spherical, planktonic. Single cup– Type locality: Lake Oloidien, Kenya. shaped chloroplast with starch–covered pyrenoid. Etymology: the species is named after its the Asexual reproduction by autosporulation, sexual locus classicus, lake Oloidien in Kenya. reproduction not observed. Cells solitary or in Authentic strain: CCAP 222/32. colonies of 2–4 cells, covered by a gelatinous Iconotype: Figure 19* envelope. Genus differs from other genera of the family by the order of the nucleotides in SSU and Kalenjinia Krienitz, C. Bock, Kotut et ITS rRNA gene sequences. Pröschold gen. nov. Typus generis: Kalenjinia gelatinosa Krienitz, Latin diagnosis: Cellulae viridis, sphaericae, C. Bock, Kotut et Pröschold sp. nov. planctonicae. Chloroplastus unicus, parietalis, Etymology: the genus is named after the Kalenjin, poculiformis, pyrenoide granis amylis tecto. Reproductio asexualis autosporum ope, reproductio a famous tribe of long distance runners in Kenya. sexualis ignota. Cellulae solitariae vel in coloniis, 2–4 cellularis, interdum tegumento gelatinoso vestitae. A Kalenjinia gelatinosa Krienitz, C. Bock, Kotut generibus ceteris familiae ordine nucleotidorum in 18S et Pröschold sp. nov. 248 Krienitz et al.: Genotypic diversity of Dictyosphaerium

Cells solitary or in 2–4 celled colonies, covered by a gelatinous envelope, planktonic. Cells connected by hyaline stalks. Cells ovoid, drop–shaped, oval or spherical, 4–14 × 3.5–9 µm. Single cup– shaped chloroplast with starch–covered pyrenoid. Asexual reproduction by autosporulation. Differs from species of other genera by the order of the nucleotides in SSU and ITS rRNA gene sequences. Holotype: material of the authentic strain CCAP 222/8 is cryopreserved in metabolic inactive state at the Culture Collection of Algae and Protozoa, Oban, Scotland. Isotype: an air–dried as well as a formaldehyde– 16 fixed sample of the authentic strain CCAP 222/8, deposited at the Botanical Museum at Berlin– Dahlem under the designation B40004043. Type locality: sewage oxidation pond, Nakuru, Kenya. Etymology: the species is named according to the gelatinous envelope which covers the alga. Authentic strain: CCAP 222/8. Iconotype: Figure 20*.

Discussion

How diverse are coccoid green algae of the Dictyosphaerium–morphotype from African inland waters based on morphology and phylogeny? According to Krienitz et al. (2004), the attachment of mucilaginous strands on either the apical or longitudinal side of the cells serves as a 17 morphological criterion that can be used to roughly differentiate between members of the Chlorella– Fig 16. Drawings of light microscopy characters of and Parachlorella–clades respectively. However, Marasphaerium gattermannii in culture. The iconotype is very limited phenotypic criteria were given to indicated by an asterisk. Scale bar 10 µm. differentiate between species and genera (see Table 3). In contrast, the results revealed a high Fig 17. Drawings of light microscopy characters of Compactochlorella kochii in culture. The iconotype is genotypic diversity of the Dictyosphaerium–like indicated by an asterisk. Scale bar 10 µm. algae. This morphotype of spherical colonial green algae evolved independently within seven different evolutionary lineages of Trebouxiophyceae. Latin diagnosis: Cellulae solitariae vel in coloniis, Within the Chlorella–clade, two species of 2–4 cellularis, interdum tegumento gelatinoso vestitae, colony–forming Hindakia, H. tetrachotoma and planctonicae. Cellulis funibus subtilis hyalinis H. fallax, were established as new combinations iunctis. Cellulae ovoides, guttae–formis, ovalis vel sphericae, 4–14 × 3.5–9 µm. Chloroplastus unicus, by Bock et al. (2010). Additionally, three solitary parietalis, poculiformis, pyrenoide granis amylis tecto. African strains were placed directly in the genus Reproductio asexualis autosporum ope. A speciebus Chlorella and given new descriptions as Chlorella ceteris generis ordine nucleotidorum in 18S rDNA et rotunda, C. singularis and C. volutis C. Bock, ITS differt. Krienitz et Pröschold (bock et al. 2011a). After Fottea, Olomouc, 12(2): 231–253, 2012 249

the description of colony–forming members of Chlorella by Bock et al. (2011a) such as C. colonialis C. Bock, Krienitz et Pröschold, we expected similar species to occur also in our samples. However, we did not find such species in our study. Based on the careful inventory provided by Huss et al. (1999), at present, we have to realize step by step that the diversity of the Chlorellaceae is much higher than expected by the authors cited. Already Müller et al. (2005) revealed by amplified fragment length polymorphism (AFLP) analyses of strains of Chlorella vulgaris Beijerinck from different international strain collections a considerable genomic divergence supporting the existence of cryptic species. In the Parachlorella–clade, six lineages had the Dictyosphaerium–morphotypes. The fact that a huge genetic diversity is hidden by a relatively uniform shape is widely reported in Trebouxiophyceae, for example, in ellipsoidal 18 Chlorella–like algae (Darienko et al. 2010) and spherical Chlorella–like algae (Bock et al. 2011a, Pröschold et al. 2011a). This high genetic diversity resulted in the description of five new species placed in four new genera in this study (see Generic and species descriptions). Furthermore, two lineages contained Dictyosphaerium–morphospecies, which were not considered in detail in this study. The strain CCAP 222/25 evolved next to the lineages of Dictyosphaerium and Parachlorella and will be studied and described by Škaloud and co–workers in near future. The strain CCAP 222/43, which is closely related to the needle–shaped Closteriopsis acicularis exhibited typical Dictyosphaerium–like 19 colonies. We did not describe this strain as a new taxon, because this enigmatic relationship of so different morphotypes needs further investigations including a wider collection of strains. Our results revealed that the highest diversity of Chlorellaceae occurred in Nakuru town sewage

Fig 18. Drawings of light microscopy characters of Compactochlorella dohrmannii in culture. The iconotype is indicated by an asterisk. Scale bar 10 µm.

Fig 19. Drawings of light microscopy characters of Masaia oloidia in culture. The iconotype is indicated by an asterisk. Scale bar 10 µm.

Fig 20. Drawings of light microscopy characters of Kalenjinia 20 gelatinosa in culture. The iconotype is indicated by an asterisk. Scale bar 10 µm. 250 Krienitz et al.: Genotypic diversity of Dictyosphaerium pond, where we found Chlorella singularis, similarity of species composition with increasing Compactochlorella dohrmannii, Masaia oloidea geographic distance. Coleman (2001) found local and Kalenjinia gelatinosa. Obviously, such adaptation and endemism in phytoflagellates of sewage ponds are hot spots for occurrence of the genera Pandorina and Volvulina. A study using the close relatives of Chlorella (Uhlmann 1966, phenotypic and genotypic criteria (Vanormelingen 1967). Kotut et al. (2010) also detected by et al. 2008) established a distribution range light microscopy Hindakia tetrachotoma (syn. for diatoms ranging from a global to a narrow Dictyosphaerium tetrachotomum Printz). In our endemic distribution range. Molecular analyses survey, we were not successful in the isolation of of a marine, picoplanktonic morphospecies– Hindakia from Nakuru sewage pond. However, complex Micromonas pusilla Butcher revealed we collected it from Lake Baringo. genotypes of global oceanic distribution and The high diversity of the Dictyosphaerium– genotypes with a more restricted distribution morphotype is also shown outside the (Slapeta et al. 2006). All these findings contradict Trebouxiophyceae. Within the Chlorophyceae the conception of universal distribution of micro– a clade evolved a cluster containing solitary organisms accentuated by Fenchel et al. (1997), and colonial species of the genus Mychonastes Finley (2002) and Fenchel & Finley (2004). (Krienitz et al. 2011). Previously, the colonial Recently, more and more arguments questioning Dictyosphaerium–like species were considered the validity of the universal distribution of micro– as members of the genus Pseudodictyosphaerium organisms have been generated (Coleman 2002; (Hindák 1988, Krienitz et al. 1999). In African Logares 2006; Foissner 2008). inland waters five different Mychonastes–species Evidently, phycogeographical conside- were found, four of them established colonies: rations and designation of phyco–floral regions M. afer, M. ovahimbae, M. racemosus and M. are of great interest. Padisák (2009) explained that timauensis Krienitz, C. Bock, Dadheech et different geographic distribution pattern of algal Pröschold (Krienitz et al. 2011). taxa are as a result of a difference in the balance between the speed of dispersal and the evolution Are the tropical Dictyosphaerium–like algae rate: If the dispersal rate is faster than the evolution genotypically identical with those from the rate, the taxon has a wide or ‘subcosmopolitan’ temperate climatic region? distribution (species occurring throughout the We did not find a simple answer to this question. world but only in appropriate habitats). In contrast, On one hand, strains from both the temperate if the rate of dispersal is slower than the rate of region and the tropics were found in one clade. evolution, floristic regionality is supported. In a For example, the strains of the type species study focusing on desmids, a morphologically well D. ehrenbergianum from UK and Germany studied algal group, Coesel (1996) established (temperate) were similar to those from Kenya the following distinct phycogeographic regions: and Tunisia (tropics). This was also the case for Indo–Malaysia/Northern Australia, Equatorial the filing strains of the members of the genus Africa, Tropical South and Central America, North Hindakia and Compactochlorella from Africa and America, Extratropical South America, Eastern Europe. In contrast to the above, the clusters of Asia, Southern Australia and New Zealand, Mucidosphaerium comprised exclusively of strains South Africa, Temperate Eurasia and finally the from Europe while those of Masaia contained circumpolar and high mountain regions. Further only East African strains. These findings are in studies should reveal the extent to which these close agreement with findings of earlier studies phycogeographic regions apply to the coccoid on the geographic distribution of algae. Using green algae and other algal groups with a high a morphological approach, Komárek (1983) level of ubiquity. compared the coccoid green algae from Cuba with species commonly found in the temperate zones. His study revealed that out of total of 109 taxa Acknowledgements We thank the Government of Kenya for permission from this tropical island, 53 taxa were identical to to carry out this research (permit number MOEST species from the temperate latitudes. The remaining 13/001/31 C 90). We are grateful to the German taxa were found exclusively in the tropics, with 21 Federal Ministry of Education and Research for taxa known only from Cuba. A study on diatoms its financial support (grant number 01LC0001). by Hillebrandt et al. (2001) revealed a decreasing Additional financial support was provided by the Fottea, Olomouc, 12(2): 231–253, 2012 251

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